Abstract
To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.
Highlights
Cellular metabolites and exogenous DNA-damaging agents constantly modify DNA
This epigenetic modification is layered on the primary genetic information and can silence the affected gene
This rupture was repaired by a very precise mechanism: the damaged chromosome pairs and retrieves genetic information from an undamaged and homologous DNA partner. This homology-directed repair was marked in half of the repaired molecules by de novo methylation of cytosines flanking the cut
Summary
Cellular metabolites and exogenous DNA-damaging agents constantly modify DNA. Such modifications may induce death in unicellular organisms or degenerative changes and aging in multicellular organisms. DNA damage can activate or amplify biochemical pathways that regulate cell growth and division or coordinate DNA replication with DNA repair. Of the various forms of DNA damage, the most hazardous to the cell are DNA double-strand breaks (DSBs). DSBs are generated when the two complementary strands of the DNA double helix are broken simultaneously at nearby sites. Dissociated ends can recombine inappropriately with other genomic sites, leading to chromosomal translocations
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